Imperial College London
Alternate

Professor Mark Isalan - Deputy Head of Department

Faculty of Natural Sciences, Department of Life Sciences

Professor of Synthetic Biology
 
 
 
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Contact

 

+44 (0)20 7594 6482m.isalan

 
 
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Location

 

509Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Citation

BibTex format

@article{Qi:2024:10.1007/978-1-0716-3718-0_1,
author = {Qi, L and Bennett, E and Isalan, M},
doi = {10.1007/978-1-0716-3718-0_1},
journal = {Methods Mol Biol},
pages = {1--13},
title = {A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display.},
url = {http://dx.doi.org/10.1007/978-1-0716-3718-0_1},
volume = {2774},
year = {2024}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, classical methods require library construction by plasmid cloning and are limited by transformation efficiencies, typically limiting library sizes to ~106-107 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >1012 members. One of these, CIS display, is a DNA-based display technique which allows high-throughput selection of biomolecules in vitro. CIS display creates the genotype-phenotype link required for selection by a DNA replication initiator protein, RepA, that binds exclusively to the template from which it has been expressed. This method has been successfully used to evolve new protein-protein interactions but has not been used before to select DNA-binding proteins, which are major components in mammalian synthetic biology. In this chapter, we describe a directed evolution method using CIS display to efficiently select functional DNA-binding proteins from pools of nonbinding proteins. The method is illustrated by enriching the minimal transcription factor Cro from a low starting frequency (1 in 109). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries.
AU  - Qi,L
AU  - Bennett,E
AU  - Isalan,M
DO  - 10.1007/978-1-0716-3718-0_1
EP  - 13
PY  - 2024///
SP  - 1
TI  - A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display.
T2  - Methods Mol Biol
UR  - http://dx.doi.org/10.1007/978-1-0716-3718-0_1
UR  - https://www.ncbi.nlm.nih.gov/pubmed/38441754
VL  - 2774
ER  -
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